Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8234—Seed-specific, e.g. embryo, endosperm

Definitions

• the present invention relates to promoters which permit a caryopsis-specific expression or suppression of genes in genetically modified plants, to methods for the tissue-specific gene expression or gene suppression in plants, expression cassettes, recombinant vectors and host cells containing such promoters, to transgenic plant cells and plants transformed with said promoters, and to methods for generating such plant cells and plants.
• promoter sequences are in the 5′-flanking region of a transcribed gene.
• individual elements of a promoter for example transcriptional enhancers
• intron sequences Kuhlemeier (1992) Plant Mol. Biol. 19: 1-14; Luehrsen (1994) The Maize Handbook, 636-638).
• a large number of promoters capable of governing the expression of transferred genes or structural genes in plants is already known.
• the most frequently used promoter is the 35S CaMV promoter (Franck et al., Cell 1 (1980), 285-294), which leads to constitutive expression of the gene introduced.
• inducible promoters are also employed, for example for wound induction (DE-A-3843628), chemical induction (Ward et al., Plant Molec. Biol. 22 (1993), 361-366) or light induction (Fluhr et al., Science 232 (1986), 1106-1112).
• stomata-specific gene expression (DE-A4207358), seed-, tuber- and fruit-specific gene expression (reviewed in Edwards and Coruzzi, Annu. Rev. Genet. 24 (1990), 275-303; DE-A-3843627), phloem-specific gene expression (Schmülling et al., Plant Cell 1 (1989), 665-470), root-nodule-specific gene expression (DE-A-3702497) or meristem-specific gene expression (Ito et al., Plant Mol. Biol. 24 (1994), 863-878).
• promoters described frequently entails disadvantages. Promoters which bring about a constitutive expression of the genes controlled by them can be employed, for example, for generating herbicide-tolerant and pathogen-resistant plants, but have the disadvantage that the products of the genes controlled by them are present in all parts of the plant, which may be undesirable, for example when the plants are intended for consumption. A negative aspect of tissue- and/or development-independent expression of a transgene can also be an undesired effect on plant development. Inducible promoters likewise entail disadvantages, since the induction conditions are typically difficult to control in the open in the case of agricultural plants.
• transgenes are very useful for introducing resistance properties into plants or modifying metabolic procedures in plants. If a transgene is to engage in defined metabolic pathways of a plant, for example if it is to produce a novel constituent or to protect against attack by pathogens, its space- and/or time-controlled expression is only possible when using an inducible and/or tissue- and/or development-specific promoter. Only this makes possible the targeted production of desired constituents in a defined developmental stage or tissue of the plant.
• tissue- and/or development-specific promoters may be advantageous over a tissue- and/or development-independent expression for the application of antisense technology, where the expression of homologous genes is to be prevented: thus, the antisense effect takes place precisely at the developmental stage at which, or in the tissue of the plant or in which, the homologous gene is also expressed.
• Starch biosynthesis genes whose gene products are expressed specifically in the storage tissue of the caryopsis, but not in vegetative tissues, have been isolated from various plant species, for example the relevant genes or cDNA clones of GBSS I. They include the waxy locus from maize (Klösgen et al. (1986) Mol. Gen. Genet. 203: 237-244), and barley (Rohde et al. (1988) Nucleic Acid Research 16, No. 14: 7185-7186), rice (Wang et al. (1990) Nucleic Acid Research 18: 5898), potato (van der Leij et al. (1991) Mol. Gen. Genet. 228: 240-248), pea (Dry et al.
• GBSS II starch-globule-band type II starch synthase
• the aim of the present invention is thus to provide means for making possible a targeted caryopsis-specific gene expression in genetically modified plants, preferably in monocots.
• the use of the means according to the invention i.e. the nucleic acid molecules, vectors, cells or plants according to the invention, makes it possible to engage, in a tissue- and/or development-specifically defined manner, in the plant's metabolism, for example in the biosynthesis of storage starch or the utilization of the caryopsis as storage or synthesis organ for starch and other reserve substances (for example polyglucans, fatty acids, modified or unmodified storage proteins or biopolymers).
• genes can be expressed specifically and at an early point in time in the caryopsis under the control of the promoter sequences according to the invention, in particular during the grain development of cereals.
• genes can be suppressed specifically and at an early point in time in the caryopsis by what are known as gene silencing strategies (cosuppression) by means of the promoter sequences according to the invention, in particular during the grain development of cereals. Cosuppression strategies using promoters have been described in detail by Vaucheret et al. (Vaucheret et al., 1998, 16(6), 651-659).
• the promoters according to the invention thus make possible, for example, the targeted modification of storage starch: to make possible the widest possible application of starch for a very wide range of industrial requirements, it is desirable to provide plants which are capable of synthesizing starches with defined properties.
• decisive properties such as solubility, gelatinization behavior, tendency to undergo retrogradation, viscosity and complex formation are determined by the amylose/amylopectin ratio, the degree of branching of the amylopectin and the derivatization of the polymers.
• a targeted modification of such properties replaces complicated methods for separating amylose and amylopectin or the expensive chemical modification of starch.
• the present invention relates to a nucleic acid molecule with the function of a caryopsis-specific promoter, which nucleic acid molecule
• the subject matter of the present invention is furthermore a nucleic acid molecule with the function of a caryopsis-specific promoter which
• nucleic acid molecule according to the invention and “promoter according to the invention” are generally used synonymously for the purposes of the present invention.
• the promoters according to the invention are those of plant genes, preferably monocots, or derived therefrom. In a further, preferred embodiment, the promoters according to the invention are suitable for expressing or suppressing genes in genetically modified plants, preferably in monocots, in particular for the expression or suppression of starch synthase genes. In this context, the promoters according to the invention can be derived from plant genes, modified by recombinant DNA techniques and/or generated synthetically.
• the promoters according to the invention can be modified for example by being combined with further cis-regulatory elements.
• the promoters according to the invention can additionally be combined with enhancer elements in order to enhance the expression of the corresponding nucleic acid molecule without however influencing its tissue-specific expression.
• Individual cis-elements (see below) of the isolated promoters can also be combined with each other to give regulatory units.
• a “promoter” is to be understood as meaning a DNA sequence comprising the regulatory portion of a gene, preferably a structural gene.
• “Regulatory portion” of a gene is to be understood as meaning that portion that determines the expression conditions of the gene.
• a regulatory portion has a sequence motif with which transcriptional factors and RNA polymerase interact and initiate transcription of the coding portion of the gene.
• the regulatory portion can comprise one or more positive regulatory elements, known as enhancers. Additionally or instead, however, it may also comprise negatively regulatory elements, known as silencers.
• a “structural gene” is generally to be understood as meaning a genetic unit of regulatory and coding portions whose gene product is generally a protein.
• the information for the primary amino acid sequence of the gene product is present in the coding portion of the structural gene, while the regulatory portion determines when, in what tissues, under what physiological conditions and in what quantities the transcript of the coding portion is formed according to whose template the gene product is synthesized.
• caryopsis specificity for the purposes of the present invention exists when the promoter according to the invention favors the expression of a gene in the caryopsis over other tissues such as, for example, mature leaves or roots and brings about a significant increase in the caryopsis, i.e. an expression rate which is increased by a factor of 2 to 5, preferably 5 to 10, in particular 10 to 100.
• caryopsis specificity can be analyzed for example by customary reporter gene experiments.
• the promoter can, for example, be linked operably to a reporter gene, such as, for example, E. coli ⁇ -glucuronidase gene (gus) in an expression cassette or in a vector for plant transformation.
• a reporter gene such as, for example, E. coli ⁇ -glucuronidase gene (gus) in an expression cassette or in a vector for plant transformation.
• This construct is then used for transforming plants.
• the ⁇ -glucuronidase (GUS) expression in the caryopsis is then determined in comparison with other tissues such as, for example, mature leaves or roots, for example as described by Martin et al. (The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS Protocols: Using the GUS genes as a Reporter of Gene Expression, Academic Press (1992), 23-43).
• caryopsis comprises in particular pericarp and endosperm. Since these tissues undergo dynamic development, the development of the endosperm, for example, into various types of cells and tissues correlates with different biochemical activities, owing to differential gene expression. Additional reference may be made to Olsen et al. (Olsen et al., 1999, Trends in Plant Science 4 (7), 253-257).
• the promoter according to the invention permits caryopsis-specific gene expression of a coding nucleotide sequence controlled by it. It constitutes an interesting alternative to known promoters since it is also capable of mediating the gene expression in the pericarp and, additionally, since it is active in the caryopsis already at a very early point in time, i.e. approximately 15-5 dap, preferably approximately 10-5 dap, in particular approximately around 5 dap.
• the promoter according to the invention allows in particular the expression of those genes whose gene products are involved in the starch metabolism of monocots, in particular wheat, to be governed efficiently.
• the promoters according to the invention can be used in many different ways. For example, they make possible the generation of transgenic plants which, owing to a modified metabolism in the caryopsis, show a qualitatively and/or quantitatively modified composition of reserves in their storage tissue, i.e. in the cereal grain.
• the present invention also relates to promoters which exhibit a functional portion of this sequence and which, in plants, bring about a caryopsis-specific expression of a coding nucleotide sequence controlled by them.
• a “functional portion” of the promoter according to the invention is to be understood as meaning, for the purposes of the present invention, those sequences which do not comprise the complete sequences of said promoters, as defined by SEQ ID No. 1 or deposited by DSM 13398, but which are truncated. Despite the truncation, “functional portions” have the caryopsis specificity according to the invention.
• Sequences comprising a functional portion of Seq. ID No. 1 preferably exhibit one or more of the segments from SEQ ID No. 1 enumerated hereinbelow: 1-26; 31-62; 68-103; 109-140; 146-240; 247-255; 260-263; 283-294; 315-329; 337-408; 414-450; 457-500; 506-519; 524-558; 568-609; 620-638; 645-655; 661-701; 728-752; 758-770; 776-792; 802-821; 827-869; 875-889; 896-928; 957-965; 974-986; 1032-1037; 1074-1106; 1114-1139; 1145-1258; 1274-1288; 1294-1323; 1330-1343; 1355-1362; 1369-1398; 1409-1448; 1454-1485; 1496-1557; 1577-1602; 1610-1643; 1663-1689; 1696-1747; 1755
• a measure for the promoter activity is, for example, the expression rate determined for a particular marker gene when under the regulatory control of the promoter according to the invention.
• suitable marker genes are the E. coli ⁇ -glucuronidase gene (gus) (Jefferson (1987) Plant Molecular Biology Reporter Vol. 5 (4): 387-405) or the green fluorescence protein gene (gfp) (Baulcombe et al., Plant J. 7 (16) (1993), 1045-1053).
• the organ or tissue specificity can be determined readily by comparison of the expression rates for said marker genes determined from individual tissues or organs of the plant.
• Functional portions of the promoter sequences comprise, for the purposes of the present invention, naturally occurring variants of the sequences according to the invention and also artificial nucleotide sequences, for example those obtained by chemical synthesis.
• a “functional portion” is to be understood as meaning in particular also natural or artificial mutations of an originally isolated promoter sequence which have the abovementioned physiological functions and features according to the invention.
• mutants encompasses substitutions, additions, deletions, exchanges and/or insertions of one or more nucleotides, in particular of suitable cis-elements, specifically as defined hereinbelow (see below).
• the scope of the present invention also extends for example to those nucleotide sequences which can be obtained by modifying the promoter sequence defined by Seq ID No. 1 or the promoter sequence deposited by DSM 13398.
• the aim of such a modification can be, for example, the generation of fragments or the insertion or repositioning of known nucleotide motifs such as, for example, restriction cleavage sites or cis-elements.
• Functional portions of the promoter sequence according to the invention in this context also comprise those promoter variants whose promoter activity is reduced or enhanced compared with the unmodified promoter (wild type).
• functional portions of the promoter sequences according to the invention are the regions identifiable by deletion analysis (cf. examples part), preferably the sequence segments 948-3139; 1006-3139; 1240-3139; 1259-3139; 1382-3139; 1486-3139; 1514-3139; 1655-3139; 1822-3139; 1887-3139; 2138-3139 and 2176-3139 of Seq ID No. 1.
• RNA polymerase II promoter activity of a eukaryotic RNA polymerase II promoter is caused by the synergistic action of various trans-active factors (DNA-binding molecules such as proteins or hormones) which bind to the various cis-regulatory DNA elements present in the promoter, generally in a region approximately 10-20 nucleotides in length.
• trans-active factors DNA-binding molecules such as proteins or hormones
• These factors interact directly or indirectly with one or more factors of the basic transcription machinery, which eventually leads to the formation of a pre-initiation complex in the vicinity of the transcription start (Drapkin et al., Current Opinion in Cell Biology 5 (1993), 469-476).
• RNA polymerase II promoters A module-light construction of the eukaryotic RNA polymerase II promoters can be assumed where the cis-elements (modules), as components of the promoter, specifically determine its activity (Tjian and Maniatis, Cell 77 (1994), 5-8).
• a minimal promoter is to be understood as meaning a DNA sequence comprising a TATA-box located approximately 20 to 30 base pairs upstream of the transcription start, or an initiator sequence (Smale and Baltimore, Cell 57 (1989), 103-113; Zawel and Reinberg, Proc. Natl. Acad. Sci. 44 (1993), 67-108; Conaway and Conaway, Annu. Rev. Biochem 62 (1993), 161-190).
• minimal promoters examples include the ⁇ 63 to +8 ⁇ 35S promoter (Frohberg, PhD thesis at the FU Berlin FB Biologie (1994)), the ⁇ 332 to +14 minimal patatin class I promoter, and the ⁇ 176 to +4 minimal PetE promoter (Pwee et al., Plant J. 3 (1993), 437-449).
• subdomains or cis-elements of the promoter according to the invention can also be identified via deletion analyses or mutageneses (Kawagoe et al., Plant J. 5(6) (1994), 885-890).
• the test for functionality of such a subdomain or cis-elements of the promoter can be effected in planta by detecting reporter gene activity in stably transformed cells.
• the present invention therefore relates to modifications of SEQ ID No. 1 obtained in particular by the di- or multimerization of subdomains or cis-elements of SEQ ID No. 1.
• an increased promoter activity compared with the wildtype is achieved by combining the promoter according to the invention with what is known as an enhancer.
• enhancers have been described in the literature, all of which generally bring about an increase in the expression in a tissue-specific manner, the tissue specificity generally being determined by the particular enhancer used (Benfey et al., Science 250 (1990), 959-966; Benfey et al., EMBO J. 8 (1989), 2195-2202; Chen et al., EMBO J. 7, (1988), 297-302; Simpson et al., Nature 323 (1986), 551-554).
• enhancers such as, for example, the PetE enhancer (Sandhu et al., Plant Mol. Biol. 37 (1998), 885-896), which do not act in a tissue-specific manner and which can therefore be placed before the promoter according to the invention as quantitative enhancer elements in order to increase expression in the caryopsis without modifying the quality or tissue specificity of the promoter according to the invention.
• synthetic enhancers can also be used; these are, for example, derived from naturally occurring enhancers and/or are obtained by combining various enhancers.
• the present invention also relates to promoters which exhibit a nucleotide sequence which hybridizes with the nucleotide sequence defined by SEQ ID No. 1 or deposited by DSM 13398, preferably under stringent conditions, and which promoters exert, in plants, a caryopsis-specific effect on the expression of a coding nucleotide sequence controlled by them.
• stringent conditions means for example hybridization conditions as they are described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). In particular, stringent hybridization takes place under the following conditions:
• Such promoters preferably have a sequence identity of at least 30%, preferably of at least 40%, preferably of at least 50%, especially preferably of at least 60%, particularly preferably of at least 70% and advantageously of at least 80%, preferably at least 90% and particularly preferably at least 95%, with the promoter sequence shown under SEQ ID No. 1 or portions thereof.
• the sequence identity of such promoter sequences is preferably determined by comparison with the nucleotide sequence shown under SEQ ID No. 1.
• the sequence identity preferably refers to the percentage of the nucleotide residues of the shorter sequence, which are identical to the nucleotide residues of the longer sequence.
• sequence identity can be determined for example by using computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit exploits the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to identify the segment with the highest sequence identity between two sequences.
• Bestfit or another sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the present invention, the parameters are preferably set in such a way that the percentage identity over the entire length of the reference sequence is calculated and that homology gaps of up to 5% of the total number of nucleotides in the reference sequence are permitted.
• Promoter sequences which, as described above, hybridize with the nucleotide sequence defined by SEQ ID No. 1 or deposited by DSM 13398 are preferably derived from plant organisms, preferably from higher plants, especially preferably from monocots, particularly preferably from Gramineae, very especially plants of the genus Triticum.
• the present invention also relates to promoters which exhibit a functional portion of the promoters according to the invention and which, in plants, bring about a caryopsis-specific expression of a coding nucleotide sequence controlled by them and which comprise one or more sequences of SEQ ID No. 2-SEQ ID No. 8.
• the promoter according to the invention exhibits all of SEQ ID No. 1 or a functional portion of the nucleotide sequence defined by SEQ ID No. 1 or deposited by DSM 13398, in particular nucleotides 948-3139; 1006-3139; 1240-3139; 1259-3139; 1382-3139; 1486-3139; 1514-3139; 1655-3139; 1822-3139; 1887-3139; 2138-3139 and 2176-3139 from SEQ ID No. 1.
• the present invention furthermore relates to expression cassettes comprising one or more promoters according to the invention.
• expression cassette is to be understood as meaning the combination of a promoter according to the invention with a nucleic acid sequence to be expressed.
• This nucleic acid sequence can be, for example, a polypeptide-encoding sequence, for example a gene which can be linked to the promoter in sense or antisense orientation.
• the nucleic acid sequence can also code a nontranslatable RNA, for example an antisense RNA or a ribozyme. These nucleic acid sequences can be used in conjunction with the promoter according to the invention to generate plants with a modified phenotype.
• the expression cassettes according to the invention can comprise a transcription termination sequence downstream of the 3′ end of the nucleic acid sequence which is linked to the promoter.
• a “transcription termination sequence” is to be understood as meaning a DNA sequence which is located at the 3′ end of a coding gene segment and which is capable of bringing about transcription termination and, if appropriate, the synthesis of a poly-A tail.
• An example of such a termination sequence is that of the octopine synthase gene. The field worker is familiar with others.
• the present invention relates to vectors comprising at least one promoter according to the invention.
• the promoter according to the invention in such a vector is linked to restriction cleavage sites or a polylinker, either of which permits integration of any sequences downstream of the promoter.
• a “polylinker” is to be understood as meaning a DNA sequence containing recognition sequences of at least one restriction enzyme, preferably of two or more restriction enzymes.
• a vector according to the invention additionally also comprises a sequence for transcription termination, for example that of the octopine synthase gene, downstream of the promoter or the polylinker.
• the present invention relates to vectors comprising expression cassettes according to the invention.
• the vectors according to the invention comprise selection markers which are suitable for identifying, and, if appropriate, selecting, cells comprising the vectors according to the invention.
• the vectors according to the invention are suitable for transforming plant cells, especially preferably for integrating foreign DNA (for example transgenes) into the plant genome.
• foreign DNA for example transgenes
• An example of such vectors are binary vectors, some of which are commercially available.
• the present invention furthermore relates to host cells which are genetically modified with a nucleic acid molecule according to the invention (i.e. promoter according to the invention), an expression cassette according to the invention or a vector according to the invention, in particular plant cells or microbial cells, for example of the genus Agrobacterium.
• a nucleic acid molecule according to the invention i.e. promoter according to the invention
• an expression cassette according to the invention i.e. promoter according to the invention
• a vector according to the invention in particular plant cells or microbial cells, for example of the genus Agrobacterium.
• “genetically modified” means that the host cell comprises a promoter according to the invention, an expression cassette according to the invention or a vector according to the invention, preferably stably integrated into the genome of the host cell, and that the promoter, or the expression cassette, has been introduced as foreign DNA into the host cell or a precursor of this cell.
• the host cells according to the invention can either be themselves the immediate product of a transformation event or else be cells derived therefrom and which comprise a promoter according to the invention or an expression cassette according to the invention.
• Suitable host cells are prokaryotic, in particular bacterial, cells or else eukaryotic cells. Eukaryotic cells can be, for example, fungal cells, in particular those of the genus Saccharomyces.
• the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention, in particular host cells of the genus Agrobacterium , for transforming plants, plant cells, plant tissues or plant parts.
• the host cells according to the invention are plant cells, termed “transgenic plant cells” hereinbelow.
• the present invention also relates to plants comprising plant cells according to the invention.
• these plants may belong to any plant species, plant genus, plant family, plant order or plant class which is commercially utilizable. They may be monocots or else dicots.
• the plants according to the invention are preferably useful plants, i.e. plants which are of agricultural, silvicultural and/or horticultural interest.
• agricultural useful plants in particular cereal species such as, for example, wheat, oats, barley, rye, maize, rice or fodder and forage grasses (such as, for example alfalfa, white clover or red clover).
• the present invention also relates to methods for generating transgenic plant cells and plants, which comprises transforming plant cells, plant tissues, plant parts or protoplasts with a nucleic acid molecule according to the invention, a vector according to the invention, an expression cassette according to the invention or with a host cell according to the invention, preferably a microorganism, growing the transformed cells, tissues, plant parts or protoplasts in a growth medium, and, when transgenic plants are generated, regenerating plants from these.
• the invention relates to the use of one or more of the nucleic acid molecules, vectors, expression cassettes or, if appropriate, host cells according to the invention for generating transgenic host cells, in particular transgenic plant cells and plants.
• the invention relates to a method for the caryopsis-specific gene expression in plants, wherein one or more of the nucleic acid molecules according to the invention is integrated stably into the genome of a plant cell, either directly or by means of one or more of the vectors, expression cassettes or host cells according to the invention, and a plant is regenerated from said plant cell.
• the invention relates to a method for the caryopsis-specific gene suppression in plants, wherein one or more of the nucleic acid molecules according to the invention is integrated stably into the genome of a plant cell, either directly or by means of one or more of the vectors, expression cassettes or host cells according to the invention, and a plant is regenerated from said plant cell, preferably by means of cosuppression.
• the plants according to the invention can be generated by methods known to the skilled worker, for example by transforming plant cells or tissue and regenerating intact plants from the transformed cells or the tissue.
• a multiplicity of techniques is available for introducing DNA into a plant host cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of the DNA by means of the biolistic approach, and other possibilities.
• DNA When DNA is injected and electroporated into plant cells, no specific requirements as such are made to the plasmids used. Simple plasmids such as, for example, pUC derivatives can be used. However, if intact plants are to be regenerated from cells transformed thus, for example the presence of a selectable marker gene is necessary.
• the Ti or Ri plasmid are used for transforming the plant cell, at least the right border, but frequently the right and left border, of the Ti and Ri plasmid T-DNA must be linked to the genes to be introduced as flanking region.
• the DNA to be introduced must be cloned into specific plasmids, viz. either into an intermediary vector or into a binary vector.
• the intermediary vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination owing to sequences which are homologous to sequences in the T-DNA.
• This Ti or Ri plasmid additionally contains the vir region, which is necessary for transferring the T-DNA.
• Intermediary vectors are not capable of replication in agrobacteria.
• the intermediary vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
• Binary vectors are capable of replicating both in E.coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187).
• the agrobacterium acting as the host cell should contain a plasmid carrying a vir region. The vir region is necessary for transferring the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed thus is used to transform plant cells.
• T-DNA for transforming plant cells has been studied intensively and described sufficiently in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.
• plant explants can expediently be cocultured together with Agrobacterium tumefaciens or Agrobacterium rhizogenes . Then, intact plants can be regenerated from the infected plant material (for example leaf sections, stem segments, roots, but also protoplasts, or plant cells grown in suspension culture) in a suitable medium which may contain antibiotics or biocides for selecting transformed cells. The plants thus obtained can then be examined for the presence of the DNA introduced.
• suitable medium which may contain antibiotics or biocides for selecting transformed cells.
• the plants thus obtained can then be examined for the presence of the DNA introduced.
• Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation have been described (cf., for example, Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim
• Suitable methods are the electrically or chemically induced DNA uptake into protoplasts, the electroporation of partially permeabilized cells, the macroinjection of DNA into inflorescences, the microinjection of DNA into microspores and proembryos, the DNA uptake by germinating pollen, and the DNA uptake into embryos by swelling (review: Potrykus, Physiol. Plant (1990), 269-273).
• the present invention furthermore also relates to the propagation material and harvested material of the plants according to the invention, which comprises plant cells according to the invention.
• the term “propagation material” extends to all those constituents of the plant which are suitable for generating progeni via the vegetative or generative route. Examples which are suitable for vegetative propagation are cuttings, callus cultures, rhizomes, root stocks or tubers. Other propagation material encompasses, for example, fruits, seeds, seedlings, protoplasts, cell cultures and the like. The propagation material is preferably tubers or seeds.
• the present invention furthermore relates to the use of promoters according to the invention, or to the promoters identified by means of the method according to the invention, for the caryopsis-specific expression of transgenes in plant cells or plants.
• the present invention relates to the use of the promoters according to the invention, or of the promoters identified by means of the method according to the invention, for the caryopsis-specific cosuppression of genes or transgenes in plant cells or plants.
• transgene is to be understood as meaning a DNA sequence which has been introduced artificially into a plant and which contains one or more of the nucleic acid molecules according to the invention.
• one of the promoters is represented by SEQ ID No. 1, consisting of 3 809 bases of the genomic sequence of the isolated gbss I subclone p11/1 such as deposited by DSM 13398. Present therein are 3 163 bases of the 5′-flanking regions and 646 bases of the coding region of GBSS I. Comparisons of the genomic sequence shown in SEQ ID No.
• AGAAA position 104 (+) AGAAA position 141 ( ⁇ ) AGAAA position 254 ( ⁇ ) AGAAA position 409 ( ⁇ ) AGAAA position 520 ( ⁇ ) AGAAA position 559 ( ⁇ ) AGAAA position 563 ( ⁇ ) AGAAA position 656 ( ⁇ ) AGAAA position 771 ( ⁇ ) AGAAA position 822 ( ⁇ ) AGAAA position 2707 ( ⁇ ) AGAAA position 2812 ( ⁇ ) AGAAA position 2819 ( ⁇ ) AGAAA position 2923 ( ⁇ ) AGAAA Q element (ZM13) position 2842 (+) AGGTCA position 2847 (+) AGGTCA
• TATCCAY motif position 2488 ( ⁇ ) TATCCAY CGACG element (AMY3, O. sativa ) position 1761 (+) CGACG position 1289 ( ⁇ ) CGACG position 1488 ( ⁇ ) CGACG position 1748 ( ⁇ ) CGACG position 932 ( ⁇ ) CGACG Root-specific DNA elements were found at the following positions: Root motif ( Triticum aestivum POX1) position 63 (+) ATATT position 278 (+) ATATT position 501 (+) ATATT position 753 (+) ATATT position 890 (+) ATATT position 277 ( ⁇ ) ATATT position 304 ( ⁇ ) ATATT position 870 ( ⁇ ) ATATT
• DNA elements involved in a hormonally regulated gene expression by ABA were found at the following positions:
• ABRE motif ( Oryza sativa em ) position 1347 (+) TACGTGTC position 1067 ( ⁇ ) TACGTGTC ABRE motif ( Triticum aestivum L. Em ) position 1930 (+) ACGTSSSC DPBF Core (CDC3) position 941 (+) ACACNNG position 951 (+) ACACNNG position 966 (+) ACACNNG position 996 (+) ACACNNG position 1010 (+) ACACNNG position 1025 (+) ACACNNG position 1107 (+) ACACNNG position 1570 (+) ACACNNG position 1603 (+) ACACNNG position 2077 (+) ACACNNG position 296 ( ⁇ ) ACACNNG
• Auxin response factor (ARF A.thaliana ) position 2984 ( ⁇ ) TGTCTC
• Auxin response factor position 2984 ( ⁇ ) TGTCTC (ARF A. thaliana ) NtBBF1 motif (roIB) position 614 (+) ACTTTA position 793 (+) ACTTTA Ethylene RE position 3022 (+) AWTTCAAA ( L.esculentum 4) position 3028 (+) AWTTCAAA
• AT-rich regions as they are known from various other promoters as enhancer elements (J. E. Sandhu, 1998, Plant Mol. Biol. 37: 885-96) are found in the promoter represented by SEQ ID No. 1 at various positions: positions 1-958, 1024-1213, 1912-1960 and 2527-3127.
• a basal DNA element which is essential for the initiation of transcription was found at position 2378. According to Nikolov (D. B. Nikolov, 1997, PNAS 94: 15-22), the point where transcription is initiated lies 25 bp downstream of the TATA box.
• SED ID No. 1 contains further, as yet unknown sequence motifs.
• One DNA motif (CCACACACTACAA) (SEQ ID NO: 11) at position 2283 shows homologies with DNA sequence segments of the barley gbss I promoter and a DNA region in the wheat puroindolin promoter (Digeon et al. (1999) Plant Mol Biol. 39: 1101-1112; Acc. No. AJ000548), which regulates expression of the GUS reporter gene in endosperm, aleuron cells and in the pericarp in rice.
• Repeats of sequence (CA) n are located at positions 948-956, 1 007-1 015 and 1 024-1 030.
• a repeating sequence motif (CTCACC) is located at positions 1 259 and 1 267.
• Two direct sequence repeats (ACGTACGT) are located at positions 1 344 and 1 349.
• Further sequence repeats (GAGAGC) are located at position 1 558, position 1 614 (CGCGTG) and 1 644 (CCCACCGG).
• a motif of the sequence (AAAC) 4 is located at position 1 887.
• the nucleic acid molecule according to the invention as shown in SEQ ID No. 1 was disclosed at the Deutsche Sammlung für Mikroorganismen und Zelikulturen (DSMZ) in Brunswick, Germany, in compliance with the provisions of the Budapest Treaty on Mar. 17, 2000 (03.17.2000) by depositing plasmid DNA: plasmid p11/1 comprising SEQ ID No. 1, deposition number DSM 13398.
• the vectors pBluescripTM II, SK(+/ ⁇ ) and KS(+/ ⁇ ) phagemid vectors (Stratagene GmbH, Heidelberg, Germany) and Lambda Fix® II/XhoI cloning vector (Stratagene GmbH, Heidelberg, Germany) were used for cloning into E.coli bacterial strains.
• the E.coli strains DH5 ⁇ (Life Technologies, Düsseldorf, Germany) and Epicurian Coli SURE® (Stratagene GmbH, Heidelberg, Germany) were used for the Bluescript vectors.
• the Epicurian Coli strain XL1-Blue MRA (Stratagene) was used for the bacteriophage vectors.
• the genomic DNA was digested partially with the restriction enzymes BamH I or Sau3A I (Life Technologies, Düsseldorf, Germany). To this end, 3 aliquots in each case comprising 100 ⁇ g genomic DNA and 150 ⁇ l of the restriction buffers were restricted for 1 h at 37° C. in a total volume of 1.5 ml with 12.5 units, 6.25 units or 3.125 units of the restriction enzyme BamH I or with 1.56 units, 0.78 units or 0.39 units of Sau3A I. Aliquots of the partially restricted DNA were then analyzed by gel electrophoresis for the degree of restriction.
• restriction enzymes were removed from the reactions by extracting once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and chloroform/isoamyl alcohol (24:1, v/v). Finally, sucrose was added to each reaction to a final concentration of 10% (w/v).
• Size fractionation of the partially restricted DNA was effected in continuous 10-40% sucrose gradients (w/v) (Sambrook et al. (1989)). Prior to application to in each case a 15 ml sucrose gradient, the individual aliquots of the partially restricted DNAs were warmed for 10 min at 68° C. and then cooled to 20° C. The gradient was centrifuged for 24 h at 20° C. and 22 000 rpm (Beckmann, Rotor SW 40). After centrifugation, the bottoms of the centrifuge tubes were pierced, and 500 ⁇ l aliquots were collected. 30 ⁇ l from the individual fractions were separated in a 0.5% agarose gel, and the size distribution of the DNA in the individual fractions was determined.
• the genomic DNA which have been restricted with BamH I or Sau3A I was ligated into the Lambda Fix® II/Xho I cloning vector following the manufacturer's instructions (Stratagene GmbH, Heidelberg, Germany).
• the ligation reaction contained: 1 ⁇ l of the Lambda Fix® II vector, 0.4 ⁇ g of genomic DNA restricted with BamH I or Sau3A I, 0.5 ⁇ l 10 ⁇ ligation buffer, 2 Weiss units T4 DNA ligase (MBI Fermentas GmbH, St. Leon-Rot, Germany); Weiss et al. (1968) J. Biol. Chem., 243: 4543-4555) in a final volume of 5 ⁇ l.
• the in vitro packaging kit “Gigapack® II Gold” by Stratagene (Stratagene GmbH, Heidelberg, Germany) was used, following the manufacturer's instructions. 1 ⁇ l of each of the ligation reactions was added to the packaging reactions; the rest was as described in the manufacturer's instructions.
• the E.coli bacterial strain XL1-Blue MRA (P2) was used for phage amplification.
• the bacteria was subsequently pelleted for 10 min at 4° C. at 2 000 rpm and the supernatant was discarded.
• the phage titer of the genomic Sau3A I or BamH I libraries were determined by counting the phage plaques. For the primary Sau3a I or BamH I libraries, phage titers of 2.2 ⁇ 10 7 pfu/ml and 1.4 ⁇ 10 7 pfu/ml, respectively, were determined. To determine the average insert sizes, 10 individual phage clones from each library were amplified, the phage DNA was isolated (Sambrook et al. 1989), and the insert sizes were determined following restriction digestion and separation by gel electrophoresis. The average insert size is approx. 15.0 kb for the BamH I library and 15.6 kb for the Sau3A I library.
• Phage clones whose genomic inserts carry sequences of the gbss I genes were identified and isolated via plaque hybridization. To screen the genomic libraries, approx. 500 000 phages from each library were plated out. The phages were plated out and the plates were lifted following standard protocols (Sambrook et al., 1989; Stratagene Lambda Fix® II Manual). DNA fragments of cDNA clones of GBSS I (Block, M.
• a 283 bp DNA fragment of the gbss I cDNA clone was labeled in a specific PCR reaction with incorporation of DIG-labeled dUTPs (Roche Diagnostics GmbH, Mannheim, Germany).
• the PCR reaction was carried out with primers positioned within the first exon of the gbss I cDNA clone (positions 146-429).
• the PCR reaction was composed as follows:
• the PCR conditions were as follows:
• the filters were prehybridized in 5 ⁇ SSC, 3% blocking reagent (Boehringer Mannheim), 0.2% sodium dodecyl sulfate (SDS), 0.1% N-laurylsarcosin and 30 ⁇ g/ml herring sperm DNA in a water bath at 65° C.
• Hybridization with the DIG-labeled DNA probes (6 ng/ml hybridization solution) was carried out overnight at 65° C. in the above-described standard hybridization buffer. All further steps of the CSPD® chemoluminescence reaction were performed following the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany).
• Positive plaques were picked out and singled out over two individual amplification and plaque filter hybridization passages.
• the DNA of the isolated positive phages were purified with the Qiagen® Lambda Kit (Qiagen GmbH, Hilden, Germany), cleaved with various restriction enzymes and, following agarose gel electrophoresis, analyzed in Southern hybridizations with the probes which have already been described.
• the genomic inserts of the positive phage clones were cleaved with various restriction enzymes.
• the resulting subfragments were cloned into bacterial vectors (pBluescriptTM II SK(+/ ⁇ ) and KS(+/ ⁇ ) phagemid vectors; Stratagene GmbH, Heidelberg, Germany).
• gbss I specific clones with 5′-upstream regulatory elements were isolated via Southern hybridizations.
• SeqLab GmbH (Göttingen) was commissioned to sequence the genomic clones of the gbss I and its 5′-upstream regulatory elements.
• the functionality of the 5′-flanking DNA regions stated in SEQ ID No. 1 were verified in transient and stable expression analyses.
• the reporter gene used was the ⁇ -glucuronidase (GUS) gene (Jefferson (1987) Plant Molecular Biology Reporter Vol.5 (4): 387-405).
• Promoter test vectors were cloned in which the coding region of the gus gene (uidA) is under the control of the 5′-flanking DNA region stated in SEQ ID No. 1 (positions 1-3 139). Cloning was performed as a transcriptional fusion.
• the uidA gene together with the nos terminator was excized from vector pCal-GUS (uidA gene under the control of the CaMV 35S promoter; Chris Warren, Stanford University, unpublished) via a partial digest and cloned behind the multiple cloning site of pBluescript (Stratagene).
• the promoter-free vector thus generated was used for the further cloning steps.
• the 5′-untranslated leader sequence of an mRNA may also affect the tissue specific expression of a gene (Rouster et al. (1998) Plant J. 15 (3): 435-40).
• the cloned promoter test vectors therefore contain this region of the GBSS I gene.
• the ⁇ -glucuronidase start codon is at the position of the GBSS I start codons.
• the starting construct of the gbss I promoter test vector carries approximately 7.5 kb of the 5′-flanking DNA region of gbss I. Cloning into the promoter-free uidA-nos vector was performed via restriction digest of plasmids p11/1 (gbss I) and potA-nos with the enzyme combinations Nco I/Xba I, NcoI/Sac I and for a partial digest with Nco I/Sal I. The 7.5 kb 5′-flanking region was subsequently truncated by different restrictions, leading to removal of DNA regions in which some of the above-described DNA elements are positioned.
• the gbss I promoter test vector was deleted in the 5′-flanking region by restrictions with the restriction enzymes stated hereinbelow. In this manner, the following deletion constructs of the gbss I promoter were cloned:
• the transient expression analyses were carried out following the biolistic transformation of various tissues (caryopses, embryos, leaves, roots) of wheat. Embryos, leaves and roots were transformed as described by Becker et al. (Plant J. (1994)5 (2): 229-307), while the biolistic transformation of the endosperm of caryopses was carried out following a modified method of Mena et al. (Plant J. (1998) 16(1), 53-42). The reporter gene activity was detected by histochemically detecting GUS activity (Jefferson (1987) Plant Molecular Biology Reporter Vol.5 (4): 387-405).
• Example 5 The promoter test vectors and deletion constructs described in Example 5 were used to generate stably transformed wheat plants:
• the transgenic plants were generated following the method of Becker et al. (Plant J. (1994) 5 (2): 229-307).
• the selection markers used were plasmids p35S-PAT (Aventis CropScience GmbH, Frankfurt) and pAct1Dneo (Müller (1992) PhD, University of Hamburg), which carry glufosinate resistance and neomycin resistance, respectively.
• the functional analysis of the gbss I promoters was carried out following regeneration of the transgenic plants and the verification of stable and complete integration of the test constructs into the wheat genome via Southern analyses.
• the reporter gene activity in the transgenic plants regenerated was studied via a histochemical GUS detection.
• Various tissues of the transgenic plants (leaves, roots, stems, endosperm, embryo, pollen) were analyzed.
• the caryopses of the plants stably transformed with the gbss I test vectors show pronounced GUS staining in the central starch endosperm.
• the GUS activity was detected even in very young caryopses in the developing endosperm.
• an activity of the gus reporter gene in the pericarp is detectable very soon after pollination, a phenomenon no longer found in older caryopses.

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